Collagen-synthetic polymer matrices prepared using a multiple step reaction

ABSTRACT

The present invention discloses collagen-synthetic polymer matrices which are prepared using a multiple step reaction. The first step of the reaction generally involves reacting collagen with a functionally activated synthetic hydrophilic polymer to form a collagen-synthetic polymer matrix. The synthetic hydrophilic polymer may be mono- or multifunctionally activated, but is preferably difunctionally activated, resulting in the formation of a crosslinked collagen matrix. The second step comprises modifying the collagen-synthetic polymer matrix according to one or more of the following methods: further crosslinking the matrix using a multifunctionally activated synthetic polymer, conjugating the matrix using a monofunctionally activated synthetic polymer, coupling biologically active molecules or glycosaminoglycans to the matrix, crosslinking the matrix using conventional chemical crosslinking agents, or modifying the collagen in the matrix by means of various chemical reactions. An optional third step may include further modification of the collagen-synthetic polymer matrix by covalently binding, for example, biologically active molecules or glycosaminoglycans to the matrix by means of available active groups on the synthetic hydrophilic polymers. Collagen-synthetic polymer matrices prepared according to the methods of the present invention have very low immunogenicity and can therefore be used to prepare biocompatible implants for use in a variety of medical applications.

CROSS-REFERENCES

This application is a continuation-in-part of U.S. application Ser. No.08/198,128, filed Feb. 17, 1994, which is a divisional of U.S.application Ser. No. 07/922,541, filed Jul. 30, 1992 and now U.S. Pat.No. 5,328,955 which is a continuation-in-part of U.S. application Ser.No. 07/433,991filed Nov. 14, 1989, and now U.S. Pat. No. 5,162,430,issued Nov. 10, 1992, which is a continuation-in-part of U.S.application Ser. No. 07/274,071, filed Nov. 21, 1988, subsequentlyabandoned, which applications and issued patents are incorporated hereinby reference in full, and to which currently pending applications weclaim priority under 35 U.S.C. § 120.

FIELD OF THE INVENTION

This invention relates to collagen-synthetic polymer matrices which areprepared using a multiple step reaction. The first step of the reactiongenerally comprises preparation of the collagen-synthetic polymer matrixby reacting collagen with a functionally activated synthetic hydrophilicpolymer. Subsequent steps involve chemical modification of thecollagen-synthetic polymer matrix by reacting the matrix with a varietyof chemical substances, depending on the desired end use application.Such collagen-synthetic polymer matrices and the methods for preparingthem are disclosed herein.

BACKGROUND OF THE INVENTION

Daniels et al., U.S. Pat. No. 3,949,073, disclosed the preparation ofsoluble collagen by dissolving tissue in aqueous acid, followed byenzymatic digestion. The resulting atelopeptide collagen is soluble, andsubstantially less immunogenic than unmodified collagen. It may beinjected into suitable locations of a subject with a fibril-formationpromoter (described as a polymerization promoter in the patent) to formfibrous collagen implants in situ, for augmenting hard or soft tissue.This material is now commercially available from Collagen Corporation(Palo Alto, Calif.) under the trademark Zydcrm® Collagen Implant.

Miyata et al., U.S. Pat. No. 4,154,559, disclosed an ophthalmic drugdelivery system comprising a chemically modified collagen thin membranecarrier.

Davis et al., U.S. Pat. No. 4,179,337, disclosed a physiologicallyactive, water-soluble polypeptide composition comprising aphysiologically active polypeptide coupled with a coupling agent topolyethylene glycol or polypropylene glycol.

Luck et al., U.S. Pat. No. 4,488,911, disclosed a method for preparingcollagen in solution (CIS), wherein native collagen is extracted fromanimal tissue in dilute aqueous acid, followed by digestion with anenzyme such as pepsin, trypsin, or Pronase® (a trademark of AmericanHoechst Corporation, Somerville, N.J.). The enzymatic digestion removesthe telopeptide portions of the collagen molecules, providing"atelopeptide" collagen in solution. The atelopeptide collagen insolution so produced is substantially nonimmunogenic, and is alsosubstantially non-crosslinked due to loss of the primary crosslinkingregions. The collagen in solution may then be precipitated by dialysisin a moderate shear environment to produce collagen fibers whichresemble native collagen fibers. The precipitated, reconstituted fibersmay additionally be crosslinked using a chemical agent (for example,aldehydes such as formaldehyde and glutaraldehyde), heat, or radiation.The resulting products are suitable for use in medical implants due totheir biocomptibility and reduced immunogenicity.

Chu, U.S. Pat. No. 4,557,764, disclosed a "second nucleation" collagenprecipitate which exhibits a desirable malleability and putty-likeconsistency. Collagen is provided in solution (e.g., at 2-4 mg/ml), anda "first nucleation product" is precipitated by rapid titration andcentrifugation. The remaining supernatant (containing the bulk of theoriginal collagen) is then decanted and allowed to stand overnight. Theprecipitated second nucleation product is collected by centrifugation.

Chu, U.S. Pat. Nos. 4,600,533; 4,655,980; 4,689,399; and 4,725,617,disclosed methods for preparing collagen membranes having high tensilestrength by compressing and drying collagen gels.

Nguyen et al., U.S. Pat. No. 4,642,117, disclosed an injectable collagenmaterial composed of reconstituted, mechanically sheared atelopeptidecollagen fibers, which are prepared by passing reconstituted collagenfibers repeatedly through a rigid mesh screen, until a substantialreduction in fiber size and size heterogeneity is achieved. Themechanically sheared fibers may be subsequently crosslinked.

Ramshaw et al., U.S. Pat. No. 4,980,403, disclosed the precipitation ofbovine collagen (types I, II, and III) from aqueous PEG solutions, wherethere is no binding between collagen and PEG.

Miyata et al., Japanese patent application 4-227265, published Aug. 17,1992, discloses a composition comprising atelopeptide collagen linked toa polyepoxy compound. The composition is injected into the body toobtain sustained skin-lifting effects.

U.S. Pat. No. 5,162,430, issued Nov. 10, 1992 to Rhee et al., andcommonly owned by the assignee of the present application, disclosescollagen-synthetic polymer conjugates and methods of covalently bindingcollagen to synthetic hydrophilic polymers. This patent furtherdisclosed binding biologically active agents to synthetic polymermolecules, then reacting with collagen to form a three-partcollagen-synthetic polymer-active agent conjugate. Commonly owned, U.S.Pat. No. 5,292,802, issued Mar. 8, 1994, discloses methods for makingtubes comprising collagen-synthetic polymer conjugates. Commonly owned,allowed U.S. application Ser. No. 07/922,541, filed Jul. 30, 1992,discloses various activated forms of polyethylene glycol and variouslinkages which can be used to produce collagen-synthetic polymerconjugates having a range of physical and chemical properties. Commonlyowned, copending U.S. application Ser. No. 07/984,933, filed Dec. 2,1992, discloses methods for coating implants with collagen-syntheticpolymer conjugates.

Commonly owned, copending U.S. application Ser. No. 08/146,843, filedNov. 3, 1993, discloses conjugates comprising various species ofglycosaminoglycan covalently bound to synthetic hydrophilic polymers,which are optionally bound to collagen as well. Commonly owned,copending U.S. application Ser. No. 08/147,227, filed Nov. 3, 1993,discloses collagen-polymer conjugates comprising chemically modifiedcollagens such as, for example, succinylated collagen or methylatedcollagen, covalently bound to synthetic hydrophilic polymers to produceoptically clear materials for use in ophthalmic or other medicalapplications.

Commonly owned U.S. application Ser. No. 08/201,860, filed Feb. 17,1994, discloses collagen-synthetic polymer conjugates prepared usingcollagens having controlled fiber size distributions, which can beobtained, for example, by manipulation of the pH of the collagen.

All publications cited above and herein are incorporated herein byreference to describe and disclose the subject matter for which it iscited.

We now disclose collagen-synthetic polymer conjugate compositionsprepared using a multiple step reaction.

DEFINITIONS

It must be noted that, as used in this specification and the appendedclaims, the singular forms "a", "an", and "the" include plural referentsunless the context clearly dictates otherwise. For example, reference to"a conjugate" includes one or more conjugate molecules, reference to "anarticle" includes one or more different types of articles known to thoseskilled in the art and reference to "the collagen" includes mixtures ofdifferent types of collagens and so forth.

Specific terminology of particular importance to the description of thepresent invention is defined below:

The term "aqueous mixture" of collagen includes liquid solutions,suspensions, dispersions, colloids, and the like, containing collagenand water.

The term "atelopeptide collagen" refers to collagens which have beenchemically treated or otherwise processed to remove the telopeptideregions, which are known to be responsible for causing an immuneresponse in humans to collagens from other animal, such as bovine,sources.

The term "available lysine residue" as used herein refers to lysine sidechains exposed on the outer surface of collagen molecules, which haveprimary amino groups capable of reacting with activated polymericglycols. The number of available lysine residues may be determined byreaction with sodium 2,4,6-trinitrobenzenesulfonate (TNBS).

The term "biologically active molecules" is used to describe moleculessuch as growth factors, cytokines, and active peptides (which may beeither naturally occurring or synthetic) which aid in the healing orregrowth of normal tissue. The function of biologically active moleculessuch as cytokines and growth factors is two-fold: 1) they can incitelocal cells to produce new tissue, or 2) they can attract cells to thesite in need of correction. As such, biologically active molecules serveto encourage "biological anchoring" of an implant within the hosttissue. Biologically active molecules useful in conjunction with thecollagen-synthetic polymer conjugates of the present invention include,but are not limited to, cytokines such as interferons (IFN), tumornecrosis factors (TNF), interleukins, colony stimulating factors (CSFs),and growth factors such as osteogenic factor extract (OFE), epidermalgrowth factor (EGF), transforming growth factor (TGF) alpha, TGF-β(including any combination of TGF-βs), TGF-β1, TGF-β2, platelet derivedgrowth factor (PDGF-AA, PDGF-AB, PDGF-BB), acidic fibroblast growthfactor (FGF), basic FGF, connective tissue activating peptides (CTAP),β-thromboglobulin, insulin-like growth factors, erythropoietin (EPO),and nerve growth factor (NGF). The term "biologically active molecules"as used herein is further intended to encompass drugs such asantibiotics, anti-inflammatories, antithrombotics, and the like.

The terms "chemically conjugated" and "conjugated" as used herein meanattached through a covalent chemical bond. In the practice of theinvention, a hydrophilic synthetic polymer and a collagen molecule maybe covalently conjugated directly to each other by means of afunctionally active group on the synthetic hydrophilic polymer, or thecollagen and synthetic polymer may be covalently conjugated using alinking radical, so that the hydrophilic synthetic polymer and collagenare each bound to the radical, but not directly to each other.

The term "collagen" as used herein refers to all forms of collagen whichcan be used as starting materials, including those which have beenrecombinantly produced, extracted from naturally occurring sources (suchas bovine corium or human placenta), processed, or otherwise modified.

The term "collagen-in-solution" or "CIS" refers to collagen in an acidicsolution having a pH of approximately 3 or less, such that the collagenis in the nonfibrillar form.

The term "collagen suspension" refers to a suspension of collagen fibersin an aqueous carrier, such as water or phosphate-buffered saline (PBS).

The term "collagen-synthetic polymer" refers to collagen chemicallyconjugated to a synthetic hydrophilic polymer, within the meaning ofthis invention. For example, "PEG-collagen" denotes a composition of theinvention wherein molecules of collagen are covalently conjugated tomolecules of polyethylene glycol (PEG).

"Crosslinked collagen" refers to a collagen composition in whichcollagen molecules are linked by covalent bonds with multifunctionallyactivated synthetic hydrophilic polymers, such as difunctionallyactivated polyethylene glycol.

The term "dehydrated" means that the material is air-dried orlyophilized to remove substantially all unbound water.

The term "difunctionally activated" refers to synthetic hydrophilicpolymers which have been chemically derivatized so as to have twofunctional groups capable of reacting with available lysine residues oncollagen molecules. The two functionally activate groups on adifunctionally activated synthetic hydrophilic polymer are generallylocated one at each end of the polymer chain. Each functionallyactivated group on a difunctionally activated synthetic hydrophilicpolymer molecule is capable of covalently binding with a collagenmolecule, thereby effecting crosslinking between the collagen molecules.

The term "effective amount" refers to the amount of a compositionrequired in order to obtain the effect desired. Thus, a "tissuegrowth-promoting amount" of a composition conraining a biologicallyactive molecule refers to the amount of biologically active moleculeneeded in order to stimulate tissue growth to a detectable degree.Tissue, in this context, includes any tissue of the body. The actualamount which is determined to be an effective amount will vary dependingon factors such as the size, condition, sex, and age of the patient, andcan be more readily determined by the caregiver.

The term "fibrillar collagen" refers to collagens in which the triplehelical molecules aggregate to form thick fibers due to intermolecularcharge interactions, such that a composition containing fibrillarcollagen will be more or less opaque.

The term "functionally activated" refers to synthetic hydrophilicpolymers which have been chemically derivatized so as to have one ormore functional group capable of reacting with available lysine residueson collagen molecules at various locations along the polymer chain.

The terms "implant" and "solid implant" refer to any semi-solid or solidobject which is intended for insertion and long- or short-term usewithin the body.

The term "in situ" as used herein means at the site of administration.

The term "in situ crosslinking" as used herein refers to crosslinking ofa collagen implant to a patient's own collagen using multifunctionallyactivated synthetic polymers, wherein one functionally activated end ofthe synthetic polymer is covalently conjugated to a collagen molecule inthe collagen implant, and the other functionally activated end of thepolymer is free to covalently bind to collagen molecules within thepatient's own tissue.

The term "molecular weight" as used herein refers to the weight averagemolecular weight of a number of molecules in any given sample, ascommonly used in the art. Thus, a sample of PEG 2000 might contain astatistical mixture of polymer molecules ranging in weight from, forexample, 1500 to 2500, with one molecule differing slightly from thenext over a range. Specification of a range of molecular weightindicates that the average molecular weight may be any value between thelimits specified, and may include molecules outside those limits. Thus,a molecular weight range of about 800 to about 20,000 indicates anaverage molecular weight of at least about 800, ranging up to about20,000.

The term "monofunctionally activated" refers to synthetic hydrophilicpolymers which have been chemically derivatized so as to have onefunctional group capable of reacting with an available lysine residue ona collagen molecule. The functionally activate group on amonofunctionally activated synthetic hydrophilic polymer is generallylocated at one end of the polymer chain. Because they can only bind toone collagen molecule at a time, monofunctionally activated synthetichydrophilic polymers are not capable of effecting crosslinking betweencollagen molecules.

The term "multifunctionally activated" refers to synthetic hydrophilicpolymers which have been chemically derivatized so as to have two ormore functional groups capable of reacting with available lysineresidues on collagen molecules at various locations along the polymerchain. Each functionally activate group on a multifunctionally activatedsynthetic hydrophilic polymer molecule is capable of covalently bindingwith a collagen molecule, thereby effecting crosslinking between thecollagen molecules. Types of multifunctionally activated hydrophilicsynthetic polymers include difunctionally activated, tetrafunctionallyactivated, and star-branched polymers.

The term "multiple step reaction" as used herein refers to a specificseries of reaction steps used to prepare, and subsequently modify, amatrix comprising collagen covalently bound to a hydrophilic syntheticpolymer. Such multiple step reactions generally comprise at least tworeaction steps, the first of which comprises covalently binding collagento a synthetic hydrophilic polymer. Subsequent (second, third, fourth,etc.) reaction steps are directed to further modification of thecollagen-synthetic polymer matrix. Such subsequent steps will varyaccording to, and are therefore determined by, the specific chemical andbiological characteristics required for the desired end use applicationof the collagen-synthetic polymer matrix.

The term "nonfibrillar collagen" refers to collagens in which the triplehelical molecules do not aggregate to form thick fibers, such that acomposition containing nonfibrillar collagen will be optically clear.

The term "optically clear" as used herein refers to an article whichtransmits at least 90% of the visible light directed at it at athickness of 1 mm.

The term "pharmaceutically acceptable fluid caxfier" refers to fluidcarders for use in injectable or implantable formulations which arebiocompatible (i.e., do not invoke an adverse response when injected orotherwise implanted within the human body) and which may be eitheraqueous, such as water or PBS, or nonaqueous, such as a biocompatibleoil.

The term "sufficient amount" as used herein is applied to the amount ofacid, base, or salt which must be added to the collagen composition inorder to achieve the desired pH and/or fiber size.

The terms "synthetic hydrophilic polymer" or "synthetic polymer" referto polymers which have been synthetically produced and which arehydrophilic, but not necessarily water-soluble. Examples of synthetichydrophilic polymers which can be used in the practice of the presentinvention are polyethylene glycol (PEG), polyoxyethylene, polymethyleneglycol, polytrimethylene glycols, polyvinylpyrrolidones,polyoxyethylene-polyoxypropylene block polymers and cop olymers, andderivatives thereof. Naturally occurring polymers such as proteins,starch, cellulose, heparin, hyaluronic acid, and derivatives thereof areexpressly excluded from the scope of this definition.

The terms "treat" and "treatment" as used herein refer to replacement,augmentation, repair, prevention, or alleviation of defects related tosoft and/or hard tissue. Additionally, "treat" and "treatment" alsorefer to the prevention, maintenance, or alleviation of disorders ordisease using a biologically active molecule coupled to or mixed withthe conjugates of the invention.

Except as otherwise defined above, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Although anymethods and materials similar or equivalent to those described hereinmay be useful in the practice or testing of the present invention, onlythe preferred methods and materials are described below. It is notintended that the invention be limited to these preferred embodiments,however. The invention is intended to have the scope defined by theattached claims.

SUMMARY OF THE INVENTION

The present invention discloses collagen-synthetic polymer matricesprepared by the process of reacting collagen with a first synthetichydrophilic polymer to form a collagen-synthetic polymer matrix, thenfurther reacting the collagen-synthetic polymer matrix with a chemicalsubstance selected from the group consisting of a second synthetichydrophilic polymer, a biologically active agent, a glycosaminoglycan orits derivatives, a chemical crosslinking agent, an esterifying agent, anamidating agent, an acylating agent, an amino acid, a peptide, orcombinations thereof.

The invention additionally discloses in detail a preferred embodiment ofthe invention wherein the first step reaction of collagen with a firstsynthetic hydrophilic polymer to form a collagen-synthetic polymermatrix is followed by a second step comprising further reacting thecollagen-synthetic polymer matrix with a second synthetic hydrophilicpolymer.

Additionally disclosed are collagen-synthetic polymer matricescontaining biologically active agents or glycosaminoglycans. Suchmatrices are prepared using a multiple step reaction, wherein the firststep comprises reacting collagen with a first synthetic hydrophilicpolymer to form a collagen-synthetic polymer matrix, a second stepcomprises further reacting the collagen-synthetic polymer matrix with asecond synthetic hydrophilic polymer, and a third step comprisescovalently binding the collagen-synthetic polymer matrix to abiologically active agent or a glycosaminoglycan or its derivatives.

The resulting compositions have low immunogenicity and, as such, can beused in a variety of medical applications, such as in drug deliverysystems or in the preparation of various formed implants.

Further disclosed are the multiple step processes for preparing thecollagen-synthetic polymer matrices described above.

One feature of the present invention is that collagen-synthetic polymermatrices can be prepared in a more controlled and reproducible mannerusing a specific sequence of reaction steps (i.e., a "multiple stepreaction").

Another feature of the present invention is that the collagen-syntheticpolymer matrices can be tailored specifically to have the physical andchemical characteristics desired for use in a variety of therapeuticapplications, depending on the specific series of reaction stepsemployed.

Yet another feature of the present invention is that collagen-syntheticpolymer matrices containing biologically active agents can be preparedin an efficient, more controlled manner, to provide a matrix whichprovides for maximum utilization of biologically active agents.

An important feature of the present invention is that thecollagen-synthetic polymer matrices can be used to coat syntheticimplants or prosthetic devices for the purpose of improving thebiocompatibility of the implant or imparting biological activity to theimplant, in the case where biologically active molecules are bound tothe collagen-synthetic polymer matrix.

Another feature of the present invention is that implantable devices canbe prepared such that biologically active agents are distributed alongthe surface of the implant, where they can exert their greatesttherapeutic effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative amounts of unreacted PEG and bound PEG as apercentage of the total amount of PEG added to the collagen forPEG-collagen matrices having a PEG concentration of 1.5 mg S-PEG per mlcollagen.

FIG. 2 shows the relative amounts of unreacted PEG and bound PEG as apercentage of the total amount of PEG added to the collagen forPEG-collagen matrices having a PEG concentration of 5.0 mg S-PEG per mlcollagen.

FIG. 3 shows the relative amounts of unreacted PEG and bound PEG as apercentage of the total amount of PEG added to the collagen forPEG-collagen matrices having a PEG concentration of 10.0 mg S-PEG per mlcollagen.

FIG. 4 shows the actual amount, in milligrams, of bound PEG found inPEG-collagen matrices prepared using a two-step reaction, compared toPEG-collagen matrices prepared using a single step reaction, formatrices having an original PEG concentration of 1.5 mg S-PEG per mlcollagen.

FIG. 5 shows the actual amount, in milligrams, of bound PEG found inPEG-collagen matrices prepared using a two-step reaction, compared toPEG-collagen matrices prepared using a single step reaction, formatrices having an original PEG concentration of 5.0 mg S-PEG per mlcollagen.

FIG. 6 shows the actual amount, in milligrams, of bound PEG found inPEG-collagen matrices prepared using a two-step reaction, compared toPEG-collagen matrices prepared using a single step reaction, formatrices having an original PEG concentration of 10.0 mg S-PEG per mlcollagen.

FIG. 7 shows gel strength in Newtons (measured using the Instron Model4202) for PEG-collagen matrices having PEG concentrations of 1.5, 5.0,and 10.0 mg S-PEG per ml collagen.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Background of the Invention

In our earlier applications, we disclosed collagen-synthetic polymerconjugate compositions containing biologically active agents such asgrowth factors. Two methods of incorporating growth factors into thecollagen-synthetic polymer conjugate compositions were disclosed inthese applications: admixing the growth factors with thecollagen-synthetic polymer conjugate, or covalently binding the growthfactors to the collagen-synthetic polymer conjugate to form a three-partcollagen-synthetic polymer-growth factor conjugate.

U.S. Pat. No. 5,162,430 disclosed two methods by which these three-partconjugates could be prepared. The first of these was by incorporatingthe factor into the collagen prior to treatment with an activatedsynthetic hydrophilic polymer. The second method comprised reacting thefactor with a molar excess of a difunctionally activated synthetichydrophilic polymer, then adding the conjugated factor to an aqueouscollagen mixture and allowing it to react to form a collagen-syntheticpolymer-growth factor conjugate.

We have since discovered that it is possible to first form a crosslinkedcollagen-synthetic polymer matrix by reacting collagen with an activatedsynthetic hydrophilic polymer, then further reacting the matrix with avariety of chemical substances, including biologically active agentssuch as growth factors, and also including, without limitation,additional synthetic hydrophilic polymers, glycosaminoglycans, otherchemical crosslinking agents, esterifying agents, amidating agents,acylating agents, antino acids, or peptides. These chemical substancescan be bound to the collagen-synthetic polymer matrix as a second step,after matrix formation, by means of either available amino groups onremaining lysine residues on the collagen in the matrix, or remainingunreacted functional groups on synthetic polymer molecules bound to thematrix. In a preferred embodiment, the collagen-synthetic polymer matrixis first formed by reacting collagen with a multifunctionally activatedsynthetic hydrophilic polymer, then the matrix is reacted with a secondfunctionally activated synthetic polymer (which may be the same ordifferent from the first polymer) to provide available functional groupsto which additional chemical substituents, such as biologically activeagents or glycosaminoglycans, can be tethered in a third-step reaction.The physical and chemical characteristics of the resultingcollagen-synthetic polymer matrix will, of course, depend on thespecific series of reactions and the types of reactants employed.

The processes of the present invention provide for the efficientproduction of collagen-synthetic polymer matrices having desiredcharacteristics, such as biologically active molecules tethered to thesurfaces of the matrix where they can exert their greatest biologicaleffect. In accordance with a preferred method for preparing thecollagen-synthetic polymer matrices of the present invention, (a) afunctionally activated hydrophilic synthetic polymer is prepared orotherwise provided, (b) collagen is covalently bound to the synthetichydrophilic polymer to provide a collagen-synthetic polymer matrix, (c)the collagen-synthetic polymer matfix is then modified by one or more ofa variety of chemical reactions, and, optionally, (d) the modifiedcollagen-polymer matrix is further altered by covalently binding, forexample, biologically active molecules or glycosaminoglycans to thematrix by means of available functional groups on the surface of themodified collagen-synthetic polymer matrix.

Activation of Synthetic Hydrophilic Polymers

A critical step in forming the collagen-synthetic polymer matrices ofthe invention involves functionalization, or activation, of thesynthetic hydrophilic polymer. The synthetic polymers useful in thepresent invention are hydrophilic, have at least one and, preferably,two or more functional groups capable of covalently bonding with thelysine residues on a collagen molecule, and are highly pure or purifiedto a highly pure state such that the polymer is or is treated to becomepharmaceutically pure so that it may be injected or implanted into ahuman patient. Most hydrophilic synthetic polymers can be renderedwater-soluble by incorporating a sufficient number of oxygen (or, lessfrequently, nitrogen) atoms available for forming hydrogen bonds inaqueous solutions. Preferred synthetic polymers are hydrophilic, but notnecessarily water-soluble.

All suitable synthetic polymers will be nontoxic, noninflammatory, andnonimmunogenic when administered subcutaneously, and will preferably beessentially nondegradable in vivo over a period of at least severalmonths. The hydrophilic synthetic polymer may increase thehydrophilicity of the conjugate, but does not render it water-soluble.The synthetic polymers can be linear or multiply branched, but aretypically not substantially crosslinked.

Although different synthetic hydrophilic synthetic polymers can be usedin connection with forming the collagen-synthetic polymer matrices ofthe invention, the synthetic polymer must be biocompatible, hydrophilic,but relatively insoluble in water, and is preferably one or more formsof derivatized polymeric glycol, preferably, polyethylene glycol (PEG),due to its known biocompatibility. Various forms of derivatized PEG areextensively used in the modification of biologically active moleculesbecause PEG can be formulated to have a wide range of solubilities andbecause it lacks toxicity, antigenicity, inununogenicity, and does nottypically interfere with the enzymatic activities and/or conformationsof peptides. Furthermore, PEG is generally non-biodegradable and iseasily excreted from most living organisms including humans.

Multifunctionally activated synthetic polymers are most preferred foruse in the present invention, with difunctionally activated polymersbeing most preferred. Multifunctionally activated polymeric glycolspreferably have an average molecular weight between about 3000 and100,000. Difunctionally activated polymeric glycols preferably have anaverage molecular weight of between about 400 to about 40,000, mostpreferably about 3000 to about 10,000. Monofunctionally activatedpolymers may also be used in the practice of the invention. However,because monofunctionally activated synthetic polymers have only oneactivated functional group, they are capable of covalently conjugatingto collagen, but not capable of forming a crosslinked network betweencollagen molecules.

Multifunctionally activated synthetic polymers can be prepared usingvarious techniques known in the art which provide functionally groups atvarious locations along the polymer. Difunctionally activated polymericglycols typically are prepared by constructing reactive hydroxy groupsat the ends of the polymer. Multifunctionally activated syntheticpolymers are capable of crosslinking the compositions of the invention,and may further be used to attach biologically active molecules to thecollagen-synthetic polymer conjugate.

Various functionalized polyethylene glycols have been used effectivelyin fields such as protein modification (see Abuchowski et al., Enzymesas Drugs, John Wiley & Sons: New York, N.Y. (1981) pp. 367-383; andDreborg et al., Crit. Rev. Therap. Drug Carder Syst. (1990) 6:315 ),peptide chemistry (see Mutter et al., The Peptides, Academic: New York,N.Y. 2:285-332; and Zalipsky et al., Int. J. Peptide Protein Res. (1987)30:740), and the synthesis of polymeric drugs (see Zalipsky et al., Eur.Polym. J. (1983) 19:1177; and Ouchi et al., J. Macromol. Sci. -Chem.(1987) A24:1011). Various types of conjugates formed by the binding offunctionally activated polyethylene glycol with specificpharmaceutically active proteins have been disclosed and found to beuseful in medical applications, in part due to the stability of suchconjugates with respect to proteolytic digestion, reducedimmunogenicity, and longer half-lives within living organisms.

One form of polyethylene glycol is monomethoxy-polyethylene glycol(mPEG), which can be activated by the addition of a compound such ascyanuric chloride, then coupled to a protein (see Abuchowski et al., J.Bio. Chem. (1977) 252:3578). Although such methods of activatingpolyethylene glycol can be used in connection with the presentinvention, they are not preferred in that the cyanuric chloride isrelatively toxic and must be completely removed from any resultingproduct in order to provide a pharmaceutically acceptable composition.

Activated forms of PEG can be made from reactants which can be purchasedcommercially. One form of activated PEG which has been found to beparticularly useful in connection with the present invention isPEG-succinate-N-hydroxysuccinimide ester (SS-PEG) (see Abuchowski etat., Cancer Biochem. Biphys. (1984) 7:175). Activated forms of PEG suchas SS-PEG react with proteins under relatively mild conditions andproduce conjugates without destroying the specific biological activityand specificity of the protein attached to the PEG. However, when suchactivated PEGs are reacted with proteins, they react and form linkagesby means of ester bonds. Although ester linkages can be used inconnection with the present invention, they are not particularlypreferred for use in formed implants intended for long-term use withinthe human body in that they undergo hydrolysis when subjected tophysiological conditions over extended periode)f time (see Dreborg etal., Crit. Rev. Therap. Drug Carrier Syst. (1990) 6:315; and Ulbrich etal., J. Makromol. Chem. (1986) 187:1131).

It is possible to link PEG to proteins via urethane linkages, therebyproviding a more stable attachment which is more resistant to hydrolyricdigestion than the ester linkages (see Zalipsky et at., Polymeric Drugand Drug Delivery Systems, Chapter 10, "Succinimidyl Carbonates ofPolyethylene Glycol" (1991 )). The stability of urethane linkages hasbeen demonstrated under physiological conditions (see Veronese et al.,Appl. Biochem. Biotechnol. (1985) 11:141; and Larwood et al., J.Labelled Compounds Radiopharm. (1984) 21:603). Another means ofattaching the PEG to a protein can be by means of a carbamate linkage(see Beauchamp et at., Anal. Biochem. (1983) 131:25; and Berger et al.,Blood (1988) 71:1641). The carbamate linkage is created by the use ofcarbonyldiimidazole-activated PEG. Although such linkages haveadvantages, the reactions are relatively slow and may take 2 to 3 daysto complete.

The various means of activating PEG described above and publicationscited in connection with the activation means are described inconnection with linking PEG to specific biologically active proteins andnot inert, biologically inactive, natural polymers such as collagen.(See Polymeric Drug and Drug Delivery Systems, Chapter 10, "SuccinimidylCarbonates of Polyethylene Glycol" (1991 ).) Such activated PEGcompounds can be used in the preparation of covalently crosslinkedconjugates of various collagens which can be used in the preparation ofa variety of formed implants for use in medical applications.

Specific Forms of Activated PEG

For use in the present invention, polyethylene glycol is modified inorder to provide functional groups on one or, preferably, two or moresites along the length of the PEG molecule, so that covalent binding canoccur between the PEG and the primary amino groups on a collagenmolecule. Some specific activated forms of PEG are shown structurallybelow, as are generalized reaction products obtained by reactingactivated forms of PEG with collagen. In Formulas 1-7, the term COLrepresents collagen. The term PEG represents polymers having therepeating structure (OCH₂ CH₂)_(n).

The first activated PEG is difunctionally activated PEG succinimidylglutarate, referred to herein as (SG-PEG). The structural formula ofthis molecule and the reaction product obtained by reacting it with acollagen are shown in Formula 1. ##STR1##

Another difunctionally activated form of PEG is referred to as PEGsuccinimidyl (S-PEG). The structural formula for this compound and thereaction product obtained by reacting it with collagen is shown inFormula 2. In any general structural formula for the compounds, thesubscript 3 is replaced with an "n". In the embodiment shown in Formula1, n=3, in that there are three repeating CH₂ groups on either side ofthe PEG. The structure in Formula 2 results in a conjugate whichincludes an "ether" linkage which is not subject to hydrolysis. This isdistinct from the conjugate shown in Formula 1, wherein an ester linkageis provided. The ester linkage is subject to hydrolysis underphysiological conditions. ##STR2##

Yet another difunctionally activated form of polyethylene glycol,wherein n=2, is shown in Formula 3, as is the conjugate formed byreacting the activated PEG with collagen. ##STR3##

Another preferred embodiment of the invention similar to the compoundsof Formulas 2 and 3 is provided when n=1. The structural formula andresulting collagen-synthetic polymer conjugate are shown in Formula 4.It is noted that this conjugate includes both an ether and a peptidelinkage. These linkages are stable under physiological conditions.##STR4##

Yet another difunctionally activated form of PEG is provided when n=0.This compound is referred to as PEG succinimidyl carbonate (SC-PEG). Thestructural formula of this compound and the conjugate formed by reactingSC-PEG with collagen is shown in Formula 5. ##STR5##

All of the activated polyethylene glycol derivatives depicted inFormulas 1-5 involve the inclusion of the succinimidyl group. However,different activating groups can be attached at sites along the length ofthe PEG molecule. For example, PEG can be derivatized to formdifunctionally activated PEG propion aldehyde (A-PEG), which is shown inFormula 6, as is the conjugate formed by the reaction of A-PEG withcollagen. The linkage shown in Formula 6 is referred to as a --(CH₂)_(n)--NH-- linkage, where n=1-10. ##STR6##

Yet another form of activated polyethylene glycol is difunctionallyactivated PEG glycidyl ether (E-PEG), which is shown in Formula 7, as isthe conjugate formed by reacting such with collagen. ##STR7##

Many of the activated forms of polyethylene glycol described above arenow available commercially from Shearwater Polymers, Huntsville, Ala.The various activated forms of polyethylene glycol and various linkageswhich can be used to produce collagen-synthetic polymer conjugateshaving a range of physical and chemical properties are described infurther detail in copending, allowed U.S. application Ser. No.07/922,541, filed Jul. 2, 1992.

The specific form of functionally activated synthetic hydrophilicpolymer used depends on the desired end use of the collagen-syntheticpolymer matrix. The type of linkage required between the collagen andthe functionally activated polyethylene glycol will depend upon whetherthe matrix is intended for long- or short-term presence within the bodyof the patient. In general, functionally activated polyethylene glycolswhich result in ether linkages are preferred for matrices intended forlong-term use because these linkages tend to be more resistant tohydrolysis than ester linkages. Polyethylene glycols which result in theweaker ester linkage should be used when it is desired to haveshort-term presence of the matrix within the body. In fact, esterlinkages are preferred for matrices intended to provide localized drugdelivery. The covalently bound drug is released from thecollagen-synthetic polymer matrix as the ester bonds are hydrolyzed.Combinations of synthetic polymers which result in different linkagescan also be employed, as described further below.

Preparation of the Collagen-Synthetic Polymer Matrix

Collagen obtained from any source may be used to prepare thecollagen-synthetic polymer matrices of the present invention. Collagenmay be extracted and purified from human or other mammalian source, ormay be recombinantly or otherwise produced. Collagen of any type may beused, including, but not limited to, types I, II, Ill, IV, or anycombination thereof, although type I is generally preferred.Atelopeptide collagen is generally preferred over telopeptide-containingcollagen because of its reduced immunogenicity. Collagens that have beenpreviously crosslinked by radiation, heat, or other chemicalcrosslinking agents such as glutaraldehyde or carbodiimide are generallynot preferred as starting materials. The collagen should be in apharmaceutically pure form such that it can be incorporated into a humanbody without generating any significant immune response.

Fibrillar collagen prepared by methods known in the an or commerciallyavailable atelopeptide fibrillar collagen compositions, such as Zyderm®I Collagen (35 mg/ml collagen concentration) or Zyderm II Collagen (65mg/ml collagen concentration), are preferred starting materials toprepare the compositions of the present invention. The collagenconcentration of the collagen suspension should generally be within therange of about 10 to about 120 mg/ml, depending on the desired end useapplication. The collagen concentration of commercially availablecollagen compositions can be decreased by mixing the collagencomposition with an appropriate amount of sterile water or phosphatebuffered saline (PBS). Conversely, to increase the collagenconcentration, the collagen composition can be concentrated bycentrifugation, then adjusted to the desired collagen concentration bymixing with an appropriate amount of sterile water or PBS.

Nonfibrillar collagens may also be used in the practice of the presentinvention. Nonfibrillar collagens for use in the present inventioninclude collagen-in-solution ("CIS") at pH 2, as well as collagens whichhave been chemically modified so as to alter the charge distribution onthe collagen molecule and, consequently, disrupt the fiber structure ofthe collagen. Such chemically modified collagens include succinylatedcollagen and methylated collagen, which may be prepared as disclosed byMiyata et al. in U.S. Pat. No. 4,164,559. Chemically modified,nonfibrillar collagens are more or less optically clear, depending onthe degree of chemical modification.

Collagens having controlled fiber size distributions, which may beprepared as described in commonly owned U.S. application Ser. No. 08/201,860, can also be used to produce the collagen-synthetic polymermatrices of the present invention.

In a general method for preparing the collagen-synthetic polymermatrices of the present invention, collagen is first reacted with asynthetic hydrophilic polymer to form a collagen-synthetic polymermatrix. Synthetic hydrophilic polymers react with primary amino groupsfound on lysine residues in collagen molecules. For example, type Icollagen contains a total of 89 lysine residues. Each of these lysineresidues contains one free (unbound) amino group. In addition, there isone primary amino group at the N-terminal of each of the three chainscomprising type I collagen. Therefore, each molecule of type I collagencontains a total of 92 (89+3) amino groups available for reaction withsynthetic hydrophilic polymers.

The reaction between collagen and the synthetic polymer is generallyperformed in a controlled manner (i.e., using a relatively low ratio ofsynthetic polymer to collagen molecules) so that the degree ofcrosslinking is limited or maximized, as desired.

The synthetic polymer is preferably a functionally activated polymericglycol and preferably is a multifunctionally activated polyethyleneglycol, most preferably, a difunctionally activated polyethylene glycol.Monofunctionally activated polymers may be used at this stage of thereaction and may in fact be preferred for use in certain embodiments ofthe invention, as described further below. However, monofunctionallyactivated polymers are only capable of conjugating single molecules ofcollagen and are therefore not capable of forming a crosslinkedcollagen-synthetic polymer network.

The concentration of activated synthetic polymer used in the first stepof the reaction will vary depending on the collagen concentration used,the type of activated polymer used (e.g., S-PEG, SG-PEG, etc.), themolecular weight of the activated polymer, and the degree ofcrosslinking or conjugation desired. For example, when reacting asuspension of collagen having a collagen concentration of approximately35 mg/ml with a difunctionally activated S-PEG, the concentration ofS-PEG used in order to achieve the controlled crosslinking desired inthe first step of the reaction is generally within the range of about 1to about 10 milligrams of difunctionally activated S-PEG per milliliterof collagen suspension. When using a suspension of collagen having acollagen concentration of approximately 65 mg/ml, the concentration ofdifunctionally activated S-PEG used in the first step of the reaction isgenerally within the range of about 1 to about 20 milligrams of S-PEGper milliliter of collagen suspension. There are generally a number ofprimary amino groups remaining on the collagen following the first stepreaction.

Chemical Modification of the Collagen-Synthetic Polymer Matrix

Subsequent (i.e., second and/or third) steps of the multiple stepreaction are largely determined by the desired end use of the resultingcomposition. However, the second step of the reaction generally involvesmodification of the remaining primary amino groups on collagen moleculesin the matrix. For example, the collagen-synthetic polymer matrix can befurther reacted with a second multifunctionally activated polymer tocreate a more highly crosslinked collagen-synthetic polymer network, orto provide a network wherein there are a number of synthetic polymermolecules having free functional groups available for furtherconjugation with, for example, biologically active agents orglycosaminoglycans. The second synthetic polymer may be of the same orof a different type than the first synthetic polymer that was used tocreate the original collagen-synthetic polymer matfix. For example, if asynthetic polymer which results in the formation of an ether linkagebetween the collagen and polymer is used in the first reaction, it maybe desirable to use a synthetic polymer which results in the formationof an ester linkage in the second reaction, or vice versa, if thecollagen-synthetic polymer matrix is intended to degrade or partiallydegrade over time, such as when the matrix is used as a drug deliverysystem.

The concentration of activated synthetic polymer required in the secondstep of the reaction is generally approximately equal to, or in excessof, the amount required to achieve complete conjugation of all of theprimary amino groups on the collagen, which will vary depending on thecollagen concentration used and the type and molecular weight ofactivated synthetic polymer used. For example, type I collagen contains92 primary amino groups per molecule and has a molecular weight ofapproximately 300,000 daltons. Theoretically, 92 molecules of activatedsynthetic polymer would be required to conjugate all of the primaryamino groups on one molecule of type I collagen. For example, whenreacting a suspension of type I collagen having a collagen concentrationof 35 mg/ml with a synthetic hydrophilic polymer having a molecularweight of 3,755 daltons, 40.3 milligrams of polymer would be requiredper milliliter of collagen to achieve (theoretically) conjugation of allthe primary amino groups on each collagen molecule, as follows: ##EQU1##Therefore, in this particular case, a synthetic polymer concentration ofat least about 40 milligrams of synthetic polymer per milliliter ofcollagen suspension (having a collagen concentration of 35 mg/ml) wouldbe used in the second reaction. (The above formula can also be used todetermine what percentage of the primary amino groups on the collagenhave been conjugated based on the amount of synthetic polymer actuallybound to the collagen, as determined, for example, by HPLC.)

Another possibility for a second reaction is to conjugate thecollagen-synthetic polymer matrix with a monofunctionally activatedsynthetic polymer. Although conjugation with a monofunctionallyactivated polymer will not increase the degree of crosslinking in thematrix, the use of a low molecular weight, monofunctionally activatedpolymer may serve to "coat" the collagen by binding with primary aminogroups that were not conjugated in the first reaction, which may resultin a collagen-synthetic polymer matrix having reduced immunogenicitycompared with previous collagen-synthetic polymer conjugatecompositions. Therefore, if an implant having extremely lowimmunogenicity is the desired goal, conjugating the crosslinkedcollagen-synthetic polymer matfix with a monofunctionally activatedsynthetic polymer should be the second step in the reaction.

The collagen-synthetic polymer matrix formed in the first step of thereaction can also be directly coupled to any biologically active agent,drug, glycosaminoglycan or glycosaminoglycan derivative, etc., that hasreactive groups, or can be chemically derivatized to have reactivegroups, that are able to bond with remaining primary amino groups on thecollagen molecules in the matrix. Coupling biologically active moleculesto the collagen-synthetic polymer matrix provides an effective sustainedrelease drug delivery system, or can serve to biologically "anchor" thecollagen-synthetic polymer matrix to host tissue. The amount ofbiologically active agent required to be therapeutically effective isdependent on the specific agent used. Coupling glycosaminoglycans ortheir derivatives to the collagen-synthetic polymer matrix results inimplant compositions having novel physical and chemical characteristics,depending on the type of glycosaminoglycan used and the relative amountsof collagen and glycosaminoglycan in the composition. Glycosaminoglycansfor use in the present invention include hyaluronic acid, chondroitinsulfate A, chondroitin sulfate C, dermatan sulfate, keratan sulfate,keratosulfate, chitin, chitosan, heparin, and derivatives thereof. Forexample, glycosaminoglycans such as heparin have unique anticoagulationproperties which make them very desirable for use in or on anyblood-contacting implant or device, such as a vascular graft orartificial organ.

The collagen-synthetic polymer matrix can also be further crosslinked bymeans of any of a number of conventional chemical crosslinking agents,including, but not limited to, glutaraldehyde, divinyl sulfone,epoxides, carbodiimides, and imidazole. The concentration of chemicalcrosslinking agent required is dependent on the specific agent beingused and the degree of crosslinking desired.

Yet another possibility for modification of the collagen-syntheticpolymer matrix is to bind amino acids or peptides to the collagen in thematrix by first coupling the amino acid or peptide to a difunctionallyactivated synthetic polymer, then reacting it with the preformedcollagen-synthetic polymer matrix. Alternatively, the amino acid orpeptide can be chemically modified to have functional groups capable ofreacting with available amino groups on collagen molecules in thematrix. Said amino acids or peptides can serve as attachment points tobind other polymers, which have been chemically defivatized to reactdirectly with amino acid moieties, to the matrix. Such polymers include,without limitation, glycosaminoglycans, poly(N-acetyl glycosamine), andpoly(alkylene oxides) such as polyethylene glycol, polypropylene oxide,polybutylene oxide, etc.

Another option is to modify the collagen in the collagen-syntheticpolymer matrix by means of various chemical reactions, such asesterification, amidation, or acylation, depending on the desired enduse of the matrix. Esterification can be accomplished by reacting thecollagen-synthetic polymer matrix with any suitable esterifying agent,such as methanol, ethanol, or butanol. Amidation can be accomplished byreacting the matrix with any suitable amidating agent, such as glutaricanhydride or succinic anhydride. Acylation can be accomplished byreacting the matrix with a suitable acylating agent, such asbenzoylchloride or butyrylchloride. Any reaction that results in analteration of the charge distribution on the collagen will cause adisruption of the collagen fiber structure, resulting in biomaterialswhich are more or less transparent, depending on the degree to which thecollagen has been chemically modified.

Commonly owned, copending U.S. application Ser. No. 08/147,227,disclosed the conjugation of synthetic hydrophilic polymers to collagenswhich had been previously chemically modified to be nonfibrillar, suchas methylated collagen or succinylated collagen. However, nonfibrillarcollagen is very viscous. Due to its high viscosity, mixing thenonfibrillar collagen with synthetic hydrophilic polymers can be ratherdifficult, resulting in a non-uniformly crosslinked collagen-syntheticpolymer matrix, which is not desirable when preparing, for example,preformed or in situ crosslinked lenticules for long-term use on theeye. Fibrillar collagen, on the other hand, is less viscous, moreelastic, generally easier to handle, and more easily mixed withsynthetic hydrophilic polymers than nonfibrillar collagen. It istherefore advantageous, when preparing an ophthalmic (or any otheroptically clear) device, to use fibrillar collagen as the startingmaterial, mix the collagen with a synthetic hydrophilic polymer to forma collagen-synthetic polymer matrix, then chemically modify theresulting collagen-synthetic polymer matrix, such as by esterificationor amidation, to produce an optically transparent implant.

In another method for preparing optically clear collagen-syntheticpolymer matrices, nonfibrillar collagen, such as CIS, having a pH ofabout 3 or less is neutralized to pH 7, then immediately reacted with amonofunctionally activated synthetic polymer to prevent fiber formationfrom occurring, resulting in the formation of an optically clearcollagen-synthetic polymer conjugate. The resulting collagen-syntheticpolymer conjugate is able to be extruded through a free gauge needlebecause it does not contain the intermolecular crosslinks obtained whena multifunctionally activated polymer is used. The clearcollagen-synthetic polymer conjugate can subsequently be crosslinkedusing a multifunctionally activated synthetic polymer, to provide anoptically clear collagen-synthetic polymer matrix.

Further (Third-Step) Modification of the Collagen-Synthetic PolymerMatrix

A collagen-synthetic polymer matrix that has been modified according toone or more of the reactions described above can be subjected to athird-step reaction in which biologically active molecules orglycosaminoglycans are covalently bound to the matrix by means of theremaining active groups on the synthetic polymer molecules that arecovalently bound to the matrix.

Commonly owned U.S. Pat. No. 5,162,430 disclosed the binding ofbiologically active agents to synthetic polymer molecules, then reactingwith collagen to form a three-part collagen-synthetic polymer-activeagent conjugate. However, when binding the biologically active agent tothe synthetic polymer, a large excess of polymer molecules had to beused in order to obtain a majority of conjugates comprising activeagent-synthetic polymer-X (where X is a free functional group on thesynthetic polymer molecule) and to reduce the possibility of obtainingactive agent-synthetic polymer-active agent conjugates, which areinactive and, furthermore, have no remaining active groups to bind tothe collagen matrix. The active agent-synthetic polymer-X conjugateswere subsequently mixed with collagen to form active agent-syntheticpolymer-collagen conjugates.

The above process was inefficient because some of the active agentsstill formed active agent-synthetic polymer-active agent conjugates.Also, it was difficult to produce matrices having high biologicalactivity, because the concentration of active agent needed to be lowrelative to the concentration of synthetic polymer in order to avoidformation of the active agent-synthetic polymer-active agent conjugates.Combining the biologically active agent, synthetic polymer, and collagenat the same time proved to be even more inefficient because of therelatively large number of active agent-synthetic polymer-active agentconjugates formed.

In a particularly preferred embodiment of the present invention,collagen is covalently bound, preferably by means of an ether linkage,to a synthetic hydrophilic polymer, which is preferably a difunctionallyactivated polyethylene glycol, to form a crosslinked collagen-syntheticpolymer matrix. The collagen-synthetic polymer matrix is furthermedified by covalently binding, preferably by means of an ester linkage,a second synthetic hydrophilic polymer, which is preferably adifunctionally activated polyethylene glycol, to the remaining primaryamino groups on the collagen. (Any unreacted synthetic polymer can bewashed off the collagen-synthetic polymer matrix at this point.) In athird step, biologically active agents (or glycosaminoglycans or theirderivatives) are covalently bound to any remaining active ends ofsynthetic polymers which are bound by one functional group to thecollagen-synthetic polymer matrix. The above process is a very efficientmethod for producing biologically active matrices in that thepossibility of obtaining active agent-synthetic polymer-active agentconjugates is avoided, because at least one end of each polymer moleculeis already bound to a collagen molecule.

USE AND ADMINISTRATION

The collagen-synthetic polymer matrices of the present invention can beused to prepare implants for use in a variety of medical applications,such as vascular grafts, artificial organs, and heart valves. In ageneral method for preparing formed implants, collagen and amultifunctionally activated synthetic polymer are mixed and cast ormolded into the desired size and shape before substantial crosslinkinghas occurred between the collagen and the polymer. The collagen andsynthetic polymer are allowed to incubate and crosslink to achieve thedesired size and shape. Once the first step of the crosslinking reactionhas been completed, the formed implant can be further crosslinked usingmultifunctionally activated synthetic polymers or conventional chemicalcrosslinking agents, conjugated using monofunctionally activatedsynthetic polymers, and/or coupled to biologically active agents. Thesecond step reaction can be accomplished by, for example, immersing theimplant in a solution of the desired agent. For example, when preparingimplants for use in contact with blood, such as vascular grafts orartificial heart valves, it may be advantageous to couple anantithrombotic agent or an agent which prevents platelet adhesion to theimplant. Conjugating biologically active agents to the implant in such amanner results in most of the biologically active agents beingdistributed along the surfaces of the implant, where they can exerttheir greatest therapeutic effect Conjugating the implant withmonofunctionally activated synthetic polymers may serve to reducethrombosis or platelet adhesion by "smoothing out" the surface of theimplant and making it less immunogenic or reactive in general.

Tubes made from the collagen-synthetic polymer matrices of the presentinvention can be used as vascular grafts or stents, or as replacementsfor any other damaged or defective nonvascular lumen within the body,such as in the reproductive or urological systems, for example, damagedfallopian tubes or ureters. Methods for making collagen-syntheticpolymer tubes for use in various applications are described further inU.S. Pat. No. 5,292,802.

The collagen-synthetic polymer matrices of the present invention canalso be used to coat synthetic implants for implantation within thebody, including, without limitation, bone and joint prostheses, coiledplatinum wires for treating aneurysms, breast implants, catheters,artificial organs, vascular grafts (such as Dacron® or Teflon® grafts)and stents, sutures, and artificial ligaments or tendons. The implant iscoated with a solution containing collagen and synthetic polymer beforesubstantial crosslinking has been achieved between the collagen and thepolymer. The collagen and synthetic polymer are allowed to crosslink onthe surface of the implant. Once the first step of the crosslinkingreaction has been completed, the implant can be further crosslinkedusing multifunctionally activated synthetic polymers or conventionalchemical crosslinking agents, conjugated using monofunctionallyactivated synthetic polymers, and/or coupled to biologically activeagents. For example, a synthetic vascular graft may first be coated witha collagen-synthetic polymer conjugate composition, which maysubsequently be coupled to antithrombotic agents, anti-platelet adhesionagents, or glycosaminoglycans having anticoagulation properties, such asheparin. In the case of a bone implant, it may be advantageous to coupleto the collagen-synthetic polymer coating on the implant bonemorphogenic proteins or osteogenic factors which promote the growth ofnew bone around the implant and/or otherwise facilitate theincorporation of the implant within the host tissue. Methods for coatingimplants with collagen-synthetic polymer conjugates are described infurther detail in copending U.S. application Ser. No. 07/984,933, filedDec. 2, 1992.

In one preferred embodiment, a mixture of collagen and a synthetichydrophilic polymer, in an mount sufficient to allow limitedcrosslinking to occur, is applied to the surface of the object to becoated before substantial crosslinking has occurred. Crosslinking of thecollagen and the synthetic polymer is allowed to occur on the surface ofthe implant, following which the implant is immersed in a solution of asecond synthetic hydrophilic polymer. In a third-step reaction, asubstance such as a biologically active agent or a glycosaminoglycan isallowed to react with remaining functional groups on synthetic polymersbound to the collagen-synthetic polymer matrix which has been coated onthe implant. This process allows for implants to be efficiently preparedsuch that biologically active agents are distributed on the surfaces ofthe implant where they can exert their greatest therapeutic effect.

For example, in a specific preferred embodiment for preparing coatedvascular grafts or stents, collagen and an activated synthetic polymerare combined in a relatively low ratio of synthetic polymer to collagenin order to form a partially crosslinked collagen-synthetic polymermatrix and, prior to the occurrence of substantial crosslinking, eastinto the shape of a tube to form a partially crosslinkedcollagen-synthetic polymer tube. The resulting tube is then fitted intothe interior of a synthetic stent, such as that formed from a metallicwire. A second partially crosslinked collagen-synthetic polymer tube isformed and placed on the outer surface of the stent.

The two collagen-synthetic polymer tubes need not have the sameproperties. For example, in a second-step reaction after forming thepartially crosslinked collagen-synthetic polymer matrix, the outer tubecould be covalently bound to a biologically active agent, such as agrowth factor such as TGF-beta, to encourage incorporation of the stentinto the surrounding tissue. The collagen-synthetic polymer matrix ofthe inner tube could be covalently bound to an antithrombogenic agent,such as heparin, to prevent blood from clotting on the inner surface ofthe stent. Accordingly, synthetic hydrophilic polymers which formdifferent types of linkages when bound to collagen could be used to formthe inner and outer tubes. For example, the collagen matrix of the outertube may be bound by means of a synthetic polymer which results in theformation of an ester linkage between the collagen and the syntheticpolymer. As such, the bonds between the collagen and the syntheticpolymer will slowly hydrolyze as the exterior surface of the stent isincorporated into the surrounding tissue over time. Conversely, thecollagen matrix of the inner tube may be bound by means of a syntheticpolymer which results in the formation of an ether linkage, as it isimportant that the inner surface of the stent remain stable andunreactive for a long period of time.

The entire collagen-synthetic polymer-coated stent structure is thenplaced or dipped in a solution of a functionally activated synthetichydrophilic polymer, which may be of the same or of a different typethan that used to form either of the two collagen-synthetic polymertubes. The interior and exterior collagen-synthetic polymer tubes arethen allowed to crosslink with each other to form a single, continuoussurface by means of the openings in the stent.

The collagen-synthetic polymer matrices of the invention can further beused specifically as localized drug delivery systems. The desiredtherapeutic agent can be coupled directly to the collagen-syntheticpolymer matrix, which can then implanted in the body at the site in needof therapy, such as a wound or tumor. The therapeutic agents will bereleased from the matrix as the covalent bonds between the agents andthe matrix are slowly broken down by enzymatic degradation. In such asituation, it may be advantageous to use a synthetic polymer whichresults in an ether linkage when forming the collagen-synthetic polymermatrix in the first reaction, so that the matrix itself is relativelystable and resistant to hydrolytic degradation. The therapeutic agentitself may be coupled to the matrix in the second reaction by means of asynthetic polymer which forms an ester linkage, resulting in acontinuous release of the agent as the ester bonds between the agent andsynthetic polymer hydrolyze over time. Alternatively, a mixture ofsynthetic polymers, some which result in the ether linkage and somewhich result in the ester linkage, can be used to couple the agents tothe matrix, so that some of the agents are released in a sustainedmanner, and some of the agents remain tethered to the matrix, remainingactive and providing a biological effect on the natural substrate forthe active site of the protein.

Optically clear collagen-synthetic polymer matrices can also be preparedby applying the multiple step reactions of the present invention. Suchmatrices may be used in a variety of ophthalmic applications, asdescribed further below, or in any therapeutic application where anoptically clear material is desirable. In one method for producing anoptically clear collagen-synthetic polymer matrix, collagen is firstreacted with a multifunctionally activated synthetic polymer to producean optically clear, crosslinked collagen-synthetic polymer matrix, whichcan be molded or otherwise shaped to form an ophthalmic implant such asa preformed lenticule or an artificial cornea or lens. Thecollagen-synthetic polymer matrix can then be chemically modified by,for example, esterification or amidation to reduce the ionicinteractions between collagen molecules, resulting in acollagen-synthetic polymer matrix which is more or less transparent,depending on the degree of chemical modification of the matrix.

Alternatively, nonfibrillar collagen, such as CIS, having a pH of about3 or less can be neutralized to pH 7, then immediately reacted with amonofunctionally activated synthetic polymer to prevent fiber formationfrom occurring, resulting in the formation of an optically clearcollagen-synthetic polymer conjugate. The resulting collagen-syntheticpolymer conjugate is able to be extruded through a fine gauge needlebecause it does not contain the intermolecular crosslinks obtained whena multifunctionally activated polymer is used. The clearcollagen-synthetic polymer conjugate can subsequently be crosslinkedusing a multifunctionally activated synthetic polymer, such as in theformation of an in situ polymerizable lenticule on the cornea of an eye.

The multiple step techniques described above to prepare optically clearcollagen-synthetic polymer matrices may also be used to preparemembranes for use in a variety of applications, such as wound healing,drug delivery, or adhesion prevention. For example, a suspension offibrillar collagen is mixed with a multifunctionally activated syntheticpolymer, then cast as a thin layer on the bottom of a flat sheetcontainer before substantial crosslinking has occurred between thecollagen and the polymer. The resulting thin membrane is further reactedwith a monofunctionally activated synthetic polymer in order to coat anyremaining reactive groups on the collagen with synthetic polymer andthen, optionally, lyophilized. Various wound healing agents, such asTGF-beta, or drugs, such as anti-inflammatories or antibiotics, may becoupled to the collagen-synthetic polymer membrane. Factors whichprevent tissue ingrowth between organs may be coupled to membranes foruse in adhesion prevention.

In an alternative method for forming membranes, collagen is firstreacted with a monofunctionally activated synthetic polymer. Theresulting collagen-synthetic polymer conjugate can be extruded to form amembrane in situ, then subsequently crosslinked using amultifunctionally activated synthetic polymer.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake the preferred embodiments of the conjugates, compositions, anddevices and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, molecularweight, etc.) but some experimental errors and deviation should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1 Preparation of PEG-Collagen Matrices Using Single StepReaction)

Samples were prepared as follows: One (1 ) milliliter of Zyderm® ICollagen (35 mg/ml collagen concentration, available from CollagenCorporation, Palo Alto, Calif.) was mixed with 1.5 mg, 5 mg, or 10 mg ofdry, difunctionally activated S-PEG (3,755 MW) by syringe-to-syringemixing, using approximately 40-50 passes to ensure that mixing wascomplete. The samples had S-PEG concentrations of 1.5, 5.0, and 10.0 mgPEG per ml collagen, respectively. The samples were incubated in theirrespective syringes at 37° C. for 16 hours. The resulting PEG-collagencrosslinked matrices were pushed out of the large end of the syringes.Each of the three 1-ml cylindrical matrices was cut in half. One-half ofeach PEG-collagen matrix was put back into its respective syringe forfurther experimentation.

Each of the three remaining 0.5-ml matrices was washed with water toremove any unreacted PEG. The water containing unreacted PEG for eachsample was retained.

The PEG-collagen matrices were then placed in 1 M NaOH at 65°-70° C. for1 hour to hydrolyze the bound PEG. Hydrolysis was performed in order tobreak the covalent bonds between the collagen and the bound PEG so thatthe amount of PEG actually bound to the collagen could subsequently bequantified by HPLC, as described in Example 2, below. The PEG wasextracted from each of the three 0.5-ml samples using CHCl₃. The CHCl₃was subsequently evaporated under N₂. The PEG residue was then dissolvedin water.

EXAMPLE 2 (Quantification of Bound PEG in PEG-Collagen Matrices PreparedUsing Single Step Reaction)

The samples containing the unreacted PEG and previously bound(hydrolyzed) PEG obtained from the PEG-collagen matrices prepared usingthe single step reaction, as described in Example 1, above, wereanalyzed in triplicate by HPLC in order to quantify the amount of PEGthat had been bound to the collagen. HPLC analysis of PEG samples wasperformed using an isocratic elution. Conditions of HPLC analysis wereas follows:

    ______________________________________                                        Column:    Waters Ultrahydrogel 250                                           Pore Size: 250 Angstroms                                                      Column Size:                                                                             7.8 mm × 30 cm                                               Exclusion Limit:                                                                         8 × 10.sup.4 daltons                                         Injection Volume:                                                                        20 μl                                                           Mobile Phase:                                                                            5 mM Sodium Acetate buffer, pH = 5.5 at                                       21° C.                                                      Flow Rate: 0.5 ml/min                                                         Pressure:  0.8 mPa                                                            Detector:  Dual Detector System, Refractive Index & UV                                   at 260 nm                                                          ______________________________________                                    

An external standard calibration curve was obtained using PEG solutionsof various concentrations. The stock solution was prepared by dissolving10.0 mg of difunctionally activated S-PEG in 1.000 ml of deionizedwater. The solution was sequentially diluted to 5.00, 2.50, 1.25, 0.625,and 0.3125 mg/ml and analyzed by HPLC. Integrating the peak at aretention time of 16 minutes, the peak area was plotted against eachconcentration of PEG standard.

FIGS. 1-3 show the relative amounts of unreacted PEG and bound PEG as apercentage of the total amount of PEG added to the collagen for thePEG-collagen matrices having S-PEG concentrations of 1.5, 5.0, and 10.0mg/ml, respectively.

As shown in FIG. 1, the sample having an S-PEG concentration of 1.5mg/ml contained no unreacted S-PEG. As shown in FIG. 2, the samplehaving an S-PEG concentration of 5.0 mg/ml contained a small amount(about 10-15%) of unreacted S-PEG. As shown in FIG. 3, the sample havingan S-PEG concentration of 10.0 mg/ml showed a slightly higher amount ofunreacted S-PEG, about 20% of the total amount of S-PEG originally addedto the collagen.

EXAMPLE 3 (Preparation of PEG-Collagen Matrices Using Two-Step Reaction)

The 0.5-ml PEG-collagen matrices which had been put back in theirsyringes were then extruded through the needle ends of their respectivesyringes to break the matrices into small pieces. The broken matriceswere placed into test tubes and 100 mg of difunctionally activatedS-PEG, dissolved in approximately 1-2 ml of PBS, was added to each ofthe three test tubes. The test tubes containing the broken PEG-Collagenmatrices and excess PEG were incubated at 37° C. for approximately 16hours.

Each of the three PEG-collagen matrices was washed with water to removeany unreacted PEG. The PEG-collagen matrices were then placed in 1M NaOHat 65°-70° C. for 1 hour to hydrolyze the bound PEG. Hydrolysis wasperformed in order to break the covalent bonds between the collagen andthe bound PEG so that the amount of PEG actually bound to the collagencould subsequently be quantified by HPLC, as described in Example 4,below. The PEG was extracted from each of the three samples using CHCl₃.The CHCl₃ was subsequently evaporated under N₂. The PEG residue fromeach sample was then dissolved in water.

EXAMPLE 4 (Quantification of Bound PEG in PEG-Collagen Matrices PreparedUsing Two-Step Reaction)

The samples containing the previously bound (hydrolyzed) PEG obtainedfrom the PEG-collagen matrices prepared using the two-step reaction, asdescribed in Example 3, above, were analyzed in triplicate by HPLC,using the same conditions described in Example 2, in order to quantifythe amount of PEG that had been bound to the collagen. FIGS. 4-6 showthe actual amounts (in milligrams) of bound PEG found in thePEG-collagen matrices prepared using the two-step reaction, compared tomatrices prepared using the single step reaction, for PEG-collagenmatrices having original S-PEG concentrations of 1.5, 5.0, and 10.0mg/ml, respectively.

Each collagen molecule contains 92 primary amino groups available forreaction with functionally activated synthetic polymers. Theoretically,therefore, 92 molecules of PEG should be able to conjugate with the 92primary amino groups residues on one collagen molecule. An S-PEGconcentration of 1.5 mg per ml of collagen represents about 3.6molecules of S-PEG per molecule of collagen. Therefore, there are stilla large number of lysine residues available for further crosslinkingwith the excess (100 mg) PEG that was added in the second step of thecrosslinking reaction. As shown in FIG. 4, the PEG-collagen matrixhaving an original S-PEG concentration of 1.5 mg/nd containedapproximately 16-17 mg of S-PEG per 0.5-ml matrix following the secondstep of the reaction, representing approximately 76-80 molecules ofS-PEG per molecule of collagen.

As shown in FIG. 7, of the three S-PEG concentrations evaluated duringthis experiment (1.5, 5.0, and 10.0 mg S-PEG per ml collagen),PEG-collagen matrices having an S-PEG concentration of 5.0 mg/ml showthe best gel strength (as measured using the Instron Model 4202),indicating that an optimum level of crosslinking between the PEG and thecollagen has been achieved at this S-PEG concentration. An S-PEGconcentration of 5.0 mg per ml of collagen represents about 12 moleculesof S-PEG per molecule of collagen. As shown in FIG. 5, the PEG-collagenmatrix having an original S-PEG concentration of 5.0 mg/ml containedapproximately 10-11 mg of S-PEG per 0.5-ml matrix following the secondstep of the crosslinking reaction, representing approximately 48-52molecules of S-PEG per molecule of collagen. Because of the tightlycrosslinked PEG-collagen network achieved at an S-PEG concentration of5.0 mg/ml, steric hindrance may prevent binding of much additional S-PEGto the collagen matrix, which would explain why the PEG-collagenmatrices having an original S-PEG concentration of 5.0 mg/ml wouldcontain a smaller amount of bound S-PEG after the second crosslinkingreaction than the matrices which had an original S-PEG concentration ofonly 1.5 mg/ml.

As shown in FIG. 6, the PEG-collagen matrix having an original S-PEGconcentration of 10.0 mg/ml contained approximately 24-25 mg of S-PEGper 0.5-ml following the second step of the crosslinking reaction,representing approximately 96-100 molecules of S-PEG per molecule ofcollagen. As shown in FIG. 7, PEG-collagen matrices having an S-PEGconcentration of 10.0 mg/ml show a gel strength approximately equal tothat of PEG-collagen matrices having an S-PEG concentration of 1.5mg/ml, and significantly less than that of PEG-collagen matrices havingan S-PEG concentration of 5.0 mg/ml. An S-PEG concentration of 10.0 mgper ml of collagen represents about 24 molecules of S-PEG per moleculeof collagen. At this S-PEG concentration, many of the difunctionallyactivated S-PEG molecules are simply conjugated to one collagen moleculeeach, thereby eliminating available crosslinking sites on the collagenmolecule and rendering the S-PEG technically monofunctional (withregards to further reaction). This phenomenon causes the creation of alooser crosslinked PEG-collagen network.

Because of this, much additional S-PEG was allowed to bind to thePEG-collagen network during the second-step crosslinking reaction, inspite of the large amount of PEG already bound. The looser networkcreated by the high original PEG concentration in the matrix preventedthe steric hindrance that was believed to have occurred with the moreoptimally crosslinked 5.0 mg/ml PEG-collagen matrix. Because many of theS-PEG molecules were bound to only one collagen molecule instead of two,there were still a large number of primary amino groups available forfurther conjugation with PEG.

Loosely crosslinked collagen-synthetic polymer networks are desirable ina variety of applications. For example, these matrices are ideal fordelivery of biologically active agents, because they contain manysynthetic polymer molecules that are bound to the collagen-syntheticpolymer matrix by only one functional group (rather than crosslinkingtwo collagen molecules by binding one collagen molecule with each of itstwo functional groups) and therefore have another functional groupavailable for binding a biologically active molecule, such as a growthfactor or other drag. Conversely, glycosaminoglycans can also be boundto the PEG-collagen matrix in such a manner.

Loosely crosslinked collagen-synthetic polymer networks prepared asdescribed above are also useful in applications where in situcrosslinking of the collagen-synthetic polymer implant to host tissue isdesired, because of the many free functional groups on the syntheticpolymer molecules that are available for binding to host collagenmolecules.

What is claimed is:
 1. A collagen-polyethylene glycol matrix prepared bythe process of:reacting collagen with a first functionally activatedpolyethylene glycol to form a collagen-polyethylene glycol matrix; andfurther reacting the collagen-polyethylene glycol matrix with a chemicalsubstance selected from the group consisting of: a second functionallyactivated polyethylene glycol, a biologically active agent, aglycosaminoglycan and its derivatives, a chemical crosslinking agent, anesterifying agent, an amidating agent, an acylating agent, an aminoacid, a peptide, and combinations thereof.
 2. The matrix of claim 1,wherein the collagen is atelopeptide fibrillar collagen.
 3. The matrixof claim 1, wherein the first functionally activated polyethylene glycolis a difunctionally activated polyethylene glycol.
 4. The matrix ofclaim 1, wherein the collagen and the first functionally activatedpolyethylene glycol are covalently bound by means of a linkage selectedfrom the group consisting of an ether linkage, an ester linkage, and aurethane linkage.
 5. The matrix of claim 1, wherein the chemicalsubstance is a second functionally activated polyethylene glycol.
 6. Thematrix of claim 5, wherein the second functionally activatedpolyethylene glycol is selected from the group consisting of adifunctionally activated polyethylene glycol and a monofunctionallyactivated polyethylene glycol.
 7. The matrix of claim 6, wherein thecollagen and the second functionally activated polyethylene glycol arecovalently bound by means of a linkage selected from the groupconsisting of an ether linkage, an ester linkage, and a urethanelinkage.
 8. A collagen-polyethylene glycol matrix prepared by theprocess of:reacting collagen with a first functionally activatedpolyethylene glycol to form a collagen-polyethylene glycol matrix; andfurther reacting the collagen-polyethylene glycol matrix with a secondfunctionally activated polyethylene glycol.
 9. The matrix of claim 8,wherein the collagen is atelopeptide fibrillar collagen.
 10. The matrixof claim 8, wherein the first functionally activated polyethylene glycolis a difunctionally activated polyethylene glycol.
 11. The matrix ofclaim 4, wherein the collagen and the first functionally activatedpolyethylene glycol are covalently bound by means of a linkage selectedfrom the group consisting of an ether linkage, an ester linkage, and aurethane linkage.
 12. The matrix of claim 11, wherein the secondfunctionally activated polyethylene glycol is selected from the groupconsisting of a difunctionally activated polyethylene glycol and amonofunctionally activated polyethylene glycol.
 13. The matrix of claim8, wherein the collagen and the second functionally activatedpolyethylene glycol are covalently bound by means of a linkage selectedfrom the group consisting of an ether linkage, an ester linkage, and aurethane linkage.
 14. A process for preparing a collagen-polyethyleneglycol matrix comprising:reacting collagen with a first functionallyactivated polyethylene glycol to form a collagen-polyethylene glycolmatrix; and further reacting the collagen-polyethylene glycol matrixwith a chemical substance selected from the group consisting of: asecond functionally activated polyethylene glycol, a biologically activeagent, a glycosaminoglycan and its derivatives, a chemical crosslinkingagent, an esterifying agent, an amidating agent, an acylating agent, anamino acid, a peptide, and combinations thereof.
 15. The process ofclaim 14, wherein the collagen is atelopeptide fibrillar collagen. 16.The process of claim 14, wherein the first functionally activatedpolyethylene glycol is a difunctionally activated polyethylene glycol.17. The process of claim 14, wherein the collagen and the firstfunctionally activated polyethylene glycol are covalently bound by meansof a linkage selected from the group consisting of an ether linkage, anester linkage, and a urethane linkage.
 18. The process of claim 14,wherein the chemical substance is a second functionally activatedpolyethylene glycol.
 19. The process of claim 18, wherein the secondfunctionally activated polyethylene glycol is selected from the groupconsisting of a difunctionally activated polyethylene glycol and amonofunctionally activated polyethylene glycol.
 20. The process of claim18, wherein the collagen and the second functionally activatedpolyethylene glycol are covalently bound by means of a linkage selectedfrom the group consisting of an ether linkage, an ester linkage, and aurethane linkage.
 21. A process for preparing a collagen-polyethyleneglycol matrix comprising:reacting collagen with a first functionallyactivated polyethylene glycol to form a collagen-polyethylene glycolmatrix; and further reacting the collagen-polyethylene glycol matrixwith a second functionally activated polyethylene glycol.
 22. Theprocess of claim 21, wherein the collagen is atelopeptide fibrillarcollagen.
 23. The process of claim 21, wherein the first functionallyactivated polyethylene glycol is a difunctionally activated polyethyleneglycol.
 24. The process of claim 21, wherein the collagen and the firstfunctionally activated polyethylene glycol are covalently bound by meansof a linkage selected from the group consisting of an ether linkage, anester linkage, and a urethane linkage.
 25. The process of claim 21,wherein the second functionally activated polyethylene glycol isselected from the group consisting of a difunctionally activatedpolyethylene glycol and a monofunctionally activated polyethyleneglycol.
 26. The process of claim 21, wherein the collagen and the secondfunctionally activated polyethylene glycol are covalently bound by meansof a linkage selected from the group consisting of an ether linkage, anester linkage, and a urethane linkage.